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Open Access Highly Accessed Methodology article

Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients

Pieter Deschaght1, Thierry De Baere1, Leen Van Simaey1, Sabine Van daele2, Frans De Baets2, Daniel De Vos3, Jean-Paul Pirnay3 and Mario Vaneechoutte1*

Author Affiliations

1 Laboratory for Bacteriology Research (LBR), Ghent University Hospital, University of Ghent, Ghent, Belgium

2 Cystic Fibrosis Centre, Department Pediatric Pulmonology, Ghent University Hospital, University of Ghent, Ghent, Belgium

3 Laboratory for Molecular and Cellular Technology (LabMCT), Burn Wound Center, Queen Astrid Military Hospital, Brussels, Belgium

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BMC Microbiology 2009, 9:244  doi:10.1186/1471-2180-9-244

Published: 29 November 2009

Abstract

Background

Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF) patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients - to mimick as closely as possible the sputa sent to routine laboratories - to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols.

Results

In our hands, all three culture methods and the bioMérieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P. aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture.

Conclusion

In this study, no difference in sensitivity could be found for the detection of P. aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format.